Time-resolved fluorometry (TRF) employing long lifetime emitting luminescent lanthanide chelates has been applied in many specific binding assays, such as e.g. immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohistochemical assays or cell based assays to measure target analytes at very low concentration. Moreover, lanthanide chelates have been used in magnetic resonance imaging (MRI) and position emission tomography (PET).
For TRF application, an optimal label has to fulfill several requirements. First, it has to be photochemical stable both in the ground state and in the excited state and it has to be kinetically and chemically stable. The excitation wavelength has to be as high as possible, preferable over 300 nm. It has to have efficient cation emission i.e. high luminescence yield (excitation coefficient×quantum yield, εΦ). The observed luminescence decay time has to be long, and the chelate has to have good water solubility. For labeling, it should have a reactive group to allow covalent attachment to a biospecific binding reactant, and the affinity and nonspecific binding properties of the labeled biomolecules have to be retained.
Although shifting of excitation wavelength has been successfully employed in scientific literature, the problem of existing technologies has been that the energy transfer from the chromophore to the lanthanide on has been relatively weak. In other words shifting of excitation wavelength to longer wavelengths has typically been accompanied by weakening of the emission intensity. A majority of the published structures are also unsuitable to be used in aqueous environment i.e. not suitable to be used as labelling reagents in bioaffinity assays.
Knapton et al., “Fluorescent Organometallic Sensors for the Detection of Chemical-Warfare-Agent Mimics”, Angew. Chem. Int. Ed. 2006, 45, 5825-5829, discloses a water-insoluble 2,4-dimethoxyphenylethynylenepyridine.
WO 2008/020113 A1 discloses luminescent lanthanide labelling reagents, e.g. 2,2′,2″,2′″-{[2-(4-isothiocyanatophenypethyl)ethylimino]bis(methylene)bis{4-[2-methoxy-4-(carboxymethoxy)phenyl]pyridine-6,2-diyl}bis(methylenenitrilo)}-tetrakis(acetato)}terbium(III) and their use.
The structures according to the present disclosure provide a general method for shifting the excitation wavelength to above about 340 nm without compromising the chelate performance, i.e. with respect to luminescence yield. The present disclosure enables the use of cheap UV LEDs as an excitation source. Currently 365 nm UV LEDs with sufficient excitation energy are available and the excitation spectrum of the chelates according to the present disclosure strongly suggests the applicability of these chelates in combination with such UV LEDs. Application of LEDs in the instrument design enables cost reduction and miniaturization of the instrument.
Also, the structures according to the disclosure offers highly luminescent water soluble chelate structures with high excitation wavelength although the chromophore(s) has substituted alkoxy substituents in both mesomeric orto- and para-positions. The adsorption properties of the chromophore moieties are decreased due to the additional hydrophilic groups near to the long aromatic π-electron containing structure. This means that these labels should not suffer from increased background although even three independent chromophores are present in the ligand structure.